The invention relates to compositions and methods for detoxification or degradation of fumonisins and related toxins. The method has broad application in agricultural biotechnology and crop agriculture and in the improvement of food grain quality.
Fungal diseases are common problems in crop agriculture. Many strides have been made against plant diseases as exemplified by the use of hybrid plants, pesticides, and improved agricultural practices. However, as any grower or home gardener can attest, the problems of fungal plant disease continue to cause difficulties in plant cultivation. Thus, there is a continuing need for new methods and materials for solving the problems caused by fungal diseases of plants.
These problems can be met through a variety of approaches. For example, the infectious organisms can be controlled through the use of agents that are selectively biocidal for the pathogens. Another method is interference with the mechanism by which the pathogen invades the host crop plant. Yet another method, in the case of pathogens that cause crop losses, is interference with the mechanism by which the pathogen causes injury to the host crop plant. In the case of pathogens that produce toxins that are undesirable to mammals or other animals that feed on the crop plants, interference with toxin production, storage, or activity can be beneficial.
Since their discovery and structural elucidation in 1988 (Bezuidenhout et al. (1988) Journal Chem. Soc., Chem. Commun. 1988:743-745), fumonisins have been recognized as a potentially serious problem in maize-fed livestock. They are linked to several animal toxicoses including leukoencephalomalacia (Marasas et al. (1988) Onderstepoort J. Vet. Res. 55:197-204; Wilson et al. (1990) American Association of Veterinary Laboratory Diagnosticians: Abstracts 33rd Annual Meeting, Denver, Colo., Madison, Wis., USA) and porcine pulmonary edema (Colvin et al. (1992) Mycopathologia 117:79-82). Fumonisins are also suspected carcinogens (Geary et al. (1971) Coord. Chem. Rev. 7:81; Gelderblom et al. (1991) Carcinogenesis 12:1247-1251; Gelderblom et al. (1992) Carcinogenesis 13:433-437). Fusarium isolates in section Liseola produce fumonisins in culture at levels from 2 to  greater than 4000 ppm (Leslie et al. (1992) Phytopathology 82:341-345). Isolates from maize (predominantly mating population A) are among the highest producers of fumonisin (Leslie et al., supra). Fumonisin levels detected in field-grown maize have fluctuated widely depending on location and growing season, but both preharvest and post-harvest surveys of field maize have indicated that the potential for high levels of fumonisins exists (Murphy et al. (1993) J. Agr. Food Chem. 41:263-266). Surveys of food and feed products have also detected fumonisin (Holcomb et al. (1993) J. Agr. Food Chem. 41:764-767; Hopmans et al. (1993) J. Agr. Food Chem. 41:1655-1658); Sydenham et al. (1991) J. Agr. Food Chem. 39:2014-2018). The etiology of Fusarium ear mold is poorly understood, although physical damage to the ear and certain environmental conditions can contribute to its occurrence (Nelson et al. (1992) Mycopathologia 117:29-36). Fusarium can be isolated from most field grown maize, even when no visible mold is present. The relationship between seedling infection and stalk and ear diseases caused by Fusarium is not clear. Genetic resistance to visible kernel mold has been identified (Gendloff et al. (1986) Phytopathology 76:684-688; Holley et al. (1989) Plant Dis. 73:578-580), but the relationship between visible mold and fumonisin production has yet to be elucidated.
Fumonisins have been shown in in vitro mammalian cell studies to inhibit sphingolipid biosynthesis through inhibition of the enzyme sphingosine N-acetyl transferase, resulting in the accumulation of the precursor sphinganine (Norred et al. (1992) Mycopathologia 117:73-78; Wang et al. (1991) Biol. Chem. 266:14486; Yoo et al. (1992) Toxicol. Appl. Pharmacol. 114:9-15; Nelson et al. (1993) Annu. Rev. Phytpathol. 31:233-252). It is likely that inhibition of this pathway accounts for at least some of fumonisin""s toxicity, and support for this comes from measures of sphinganine:sphingosine ratios in animals fed purified fumonisin (Wang et al. (1992) J. Nutr. 122:1706-1716). Fumonisins also affect plant cell growth (Abbas et al. (1992) Weed Technol. 6:548-552; Van Asch et al. (1992) Phytopathology 82:1330-1332; Vesonder et al. (1992) Arch. Environ. Contam. Toxicol. 23:464-467). Kuti et al. (1993) (Abstract, Annual Meeting American Phytopathological Society, Memphis, Tenn.: APS Press) reported on the ability of exogenously added fumonisins to accelerate disease development and increase sporulation of Fusarium moniliform and F. oxysporum on tomato.
Enzymes that degrade the fungal toxin fumonisin to the compound AP1 have been identified in U.S. Pat. No. 5,716,820 and pending U.S. patent application Ser. Nos. 08/888,949 and 08/888,950, both filed Jul. 7, 1997, and hereby incorporated by reference. Plants expressing a fumonisin esterase enzyme, infected by fumonisin producing fungus, and tested for fumonisin and AP1 were found to have low levels of fumonisin but high levels of AP1. AP1 is less toxic than fumonisin to plants and probably also animals, but contamination with AP1 is still a concern. The best result would be complete detoxification of fumonisin to a non-toxic form. Therefore additional enzymes capable of degrading fumonisin and fumonisin catabolic products are necessary for the complete detoxification of fumonisin.
A fumonisin detoxification gene cluster is provided. Particularly, the nucleotide sequence for the cluster from Bacterium 2412.1 (American Type Culture Collection Deposit Number 55552) is provided. At least twelve structural genes and three regulatory gene are provided. The sequences represent a catabolic pathway for fumonisins and fumonisin-like compounds.
Compositions and methods for catabolism and detoxification of fumonisin, fumonisin-related toxins, and fumonisin-degradation products are provided. In particular, proteins involved in catabolism and transmembrane transport of fumonisin and fumonisin catabolic products are provided. Nucleotide sequences corresponding to the proteins are also included. The compositions are useful in the complete detoxification and degradation of fumonisin. The nucleotide sequences can be used in expression cassettes for transformation of host cells of interest. The compositions and methods of the invention provide a catabolic pathway for fumonisin. Thus, organisms can be genetically modified to provide for the complete catabolism and detoxification of fumonisin and fumonisin-related toxins.
In particular, expression cassettes for expression of the sequences in plants and other organisms are provided as well as transformed plants and other host cells.